WO2008080367A1 - Liposome formulation and process for preparation thereof - Google Patents

Liposome formulation and process for preparation thereof Download PDF

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Publication number
WO2008080367A1
WO2008080367A1 PCT/CN2007/071403 CN2007071403W WO2008080367A1 WO 2008080367 A1 WO2008080367 A1 WO 2008080367A1 CN 2007071403 W CN2007071403 W CN 2007071403W WO 2008080367 A1 WO2008080367 A1 WO 2008080367A1
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WIPO (PCT)
Prior art keywords
mol
liposome
drug
preparation according
phospholipid
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PCT/CN2007/071403
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English (en)
French (fr)
Inventor
Chunlei Li
Jinxu Wang
Caixia Wang
Yanhui Li
Dongmin Shen
Wenmin Guo
Li Zhang
Lan Zhang
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Shijiazhuang Pharma Group Zhongqi Pharmaceutical Technology Co Ltd
Original Assignee
Shijiazhuang Pharma Group Zhongqi Pharmaceutical Technology Co Ltd
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Priority to BRPI0720733-6A priority Critical patent/BRPI0720733A2/pt
Priority to MX2009007089A priority patent/MX2009007089A/es
Priority to KR1020097015712A priority patent/KR101126629B1/ko
Priority to AU2007341803A priority patent/AU2007341803B2/en
Priority to ES07846229T priority patent/ES2401526T3/es
Priority to CA2673924A priority patent/CA2673924C/en
Priority to EP07846229A priority patent/EP2123260B1/en
Priority to HK10100560.8A priority patent/HK1133398B/zh
Priority to US12/521,357 priority patent/US20110002977A1/en
Priority to JP2009543335A priority patent/JP2010514708A/ja
Application filed by Shijiazhuang Pharma Group Zhongqi Pharmaceutical Technology Co Ltd filed Critical Shijiazhuang Pharma Group Zhongqi Pharmaceutical Technology Co Ltd
Priority to RU2009126983/15A priority patent/RU2494729C2/ru
Publication of WO2008080367A1 publication Critical patent/WO2008080367A1/zh
Priority to CU2009000114A priority patent/CU23980B1/es
Anticipated expiration legal-status Critical
Priority to US15/138,527 priority patent/US10028913B2/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Liposomal pharmaceutical preparation and preparation method thereof Liposomal pharmaceutical preparation and preparation method thereof
  • the present invention relates to a liposome preparation and a liposome pharmaceutical preparation in which a drug is encapsulated, particularly a liposome pharmaceutical preparation involving mitoxantrone.
  • the invention further relates to a process for the preparation of the liposome, liposome pharmaceutical formulation and use thereof.
  • Liposomes can be used as carriers for many drugs, especially as anti-tumor drugs (especially chemotherapeutic drugs), which can reduce the distribution of drugs in normal tissues, increase the accumulation of drugs in tumor tissues, and thus improve the drug. Treatment index.
  • the reason why liposomes can passively target tumors is related to the physiological characteristics of tumors. Due to the rapid growth of tumors, the intervascular space can reach 100-780 nm, and the gap between normal vascular endothelial cells is usually around 2 nm. Therefore, if the liposome can circulate in the blood for a long period of time, and the liposome has a particle size of ⁇ 200 nm, it can be passively accumulated in the tumor area. This is because: After intravenous injection, small-sized liposomes cannot enter normal tissues during blood circulation, but can effectively penetrate blood vessels in the tumor area and reach the treatment site.
  • DMPC dimyristoyl lecithin
  • the prescription may be unstable (Liposomemal formulations of mitoxantrone, US5,858,397)»
  • Neopharm in the United States uses another technology to develop mitoxantrone liposomes. They added a negatively charged cardiolipin to the bilayer of the phospholipid. Since cardiolipin can interact strongly with mitoxantrone, mitoxantrone can be inserted into the phospholipid bilayer by passive drug loading. This technology differs from active drug loading technology. Using active drug loading techniques, the drug will be deposited as a precipitate in the internal aqueous phase of the liposome. The company's products have been discovered through a phase of clinical research. Liposomal drugs can increase the chance of sporadic infections compared to free drugs. For safety reasons, the product was discontinued (Lithoquinone liposomal formulation, CN01817424.8).
  • liposome formulations that are capable of delivering drugs with good targeting while being effectively released in target tissues, as well as corresponding liposome pharmaceutical formulations.
  • the present invention provides a liposome preparation having a particle size of about 30-80 nm and containing a phospholipid having a Tm higher than body temperature in the phospholipid bilayer, whereby the phase transition temperature of the liposome is higher than the body temperature .
  • the phospholipid include, for example, phosphatidylcholine, hydrogenated soy lecithin (HSPC), hydrogenated egg yolk lecithin, bis-lipoic lecithin (DPPC) Or bis-stearic acid lecithin (DSPC) or any combination thereof, but is not limited thereto.
  • the phospholipid having a Tm higher than body temperature in the phospholipid bilayer comprises from 50 to 100 mol/mol%, preferably from 55 to 95 mol/mol%, more preferably from 55 to 95 mol/mol%, of the total phospholipid content.
  • phospholipids in the phospholipid bilayer of the liposome preparation of the present invention for example, phospholipids having a Tm value not higher than body temperature, examples of which are dimyristoyl lecithin (DMPC), etc. .
  • DMPC dimyristoyl lecithin
  • the content of such a phospholipid in the liposome preparation of the present invention it can be conventionally determined by a person skilled in the art as long as it does not significantly lower the Tm value of the liposome preparation to below the body temperature.
  • cholesterol may optionally be contained in order to adjust the fluidity of the liposome membrane.
  • the liposome preparations of the present invention may optionally contain other excipients, particularly excipients which may further modify the surface characteristics of the liposomes, thereby providing better in vivo behavior of the liposomes.
  • excipients include, for example, lipid substances modified with a hydrophilic polymer and the like.
  • the present invention provides a liposome pharmaceutical preparation comprising a drug of interest, particularly a multivalent ion drug, in the liposome preparation of the present invention.
  • the present invention relates to a liposome pharmaceutical preparation having a particle size of between 30 and 80 nm, and: 1) containing a multivalent ion drug as an active ingredient; 2) containing a Tm higher than body temperature in the phospholipid bilayer Phospholipids such that the phase transition temperature of the liposomes is above body temperature; and optionally 3) excipients that are acceptable for other drugs and/or other liposome pharmaceutical formulations.
  • the main peak of the particle size of the liposome pharmaceutical preparation concentrates around 35-75 nm, especially about 40-60 nm.
  • a further aspect of the present invention provides a method for preparing the above liposome pharmaceutical preparation, which comprises the steps of: (1) preparing a liposome using a phospholipid having a Tm value higher than body temperature and optionally other phospholipids and/or cholesterol; (2) Encapsulating the drug of interest, particularly a multivalent ion drug, in the liposome.
  • the present invention also provides a method of treating a disease comprising administering a liposome pharmaceutical preparation of the present invention to a subject in need thereof.
  • the subject is a mammal, especially a human.
  • FIG. 1 Pharmacokinetic behavior of PLM 60 in Kunming mice and its comparison with free mitoxantrone.
  • PLM represents PEGylated mitoxantrone liposomes
  • FM is mitoxantrone free drug
  • the abscissa is time in hours.
  • the ordinate is the plasma concentration of mitoxantrone in ug mitoxindole / mL plasma.
  • FIG. 2 Distribution of PLM 60 and FM in mouse tumors.
  • PLM 60 is a PEGylated mitoxantrone liposome
  • FM is a mitoxantrone free drug
  • the abscissa is time, and the unit is hour.
  • the ordinate is the concentration of mitoxantrone in the tumor tissue, and the unit is u mitox / g tumor tissue.
  • Figure 3 Pharmacokinetic comparison of different formulations in mice.
  • the abscissa is time, the unit is hour, and the ordinate is the plasma concentration of mitoxantrone in mitoxantrone/mL plasma.
  • Different prescription doses were 4 mg/kg.
  • the liposome is composed of a membrane such as phospholipid or cholesterol. These two components not only form the basic substance of the liposome bilayer, but also have extremely important physiological functions.
  • the physical properties of liposome membranes are closely related to the temperature of the medium.
  • the acyl side chain of the lipid bilayer changes from ordered to disordered. This change causes a series of changes in the physical properties of the lipid film, which can be changed from a "gelatin” state to a "liquid crystal” state.
  • the cross-section of the film increases, the thickness of the bilayer decreases, and the fluidity of the film increases.
  • the temperature at this transition is called the phase transition temperature.
  • the phase transition temperature of the lipid film can be measured by means of Differential Scanning Calorimertry, Electron Spins Resonance (ESR), etc.
  • ESR Electron Spins Resonance
  • phase transition temperature such as the phase transition temperature of dimyristoyl cholesteryl is 24 ° C
  • dipalmitate Phosphatidylcholine and distearoylphosphatidylcholine were 41 ° C and 58 ° C, respectively.
  • the fluidity of the membrane is an important physical property of the liposome. At the phase transition temperature, the fluidity of the membrane increases, and the drug encapsulated in the liposome has the maximum release rate, thus the membrane Fluidity directly affects the stability of liposomes.
  • the invention provides a liposome formulation having a particle size of about
  • the liposome pharmaceutical preparation of the present invention is made of a phospholipid having a higher phase transition temperature Tm, such as phosphatidylcholine.
  • Tm phase transition temperature
  • the length of the hydrocarbon chain is preferably not less than 16 carbons.
  • the phospholipids of the invention include, but are not limited to, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, bis-sucrose lecithin (DPPC) or distearate lecithin (DSPC), or any combination thereof.
  • the phospholipid having a Tm higher than body temperature in the phospholipid bilayer comprises from about 50 to 100 mol/mol%, preferably from about 55 to 95 mol/mol%, more preferably from about 55 to 95 mol/mol%. About 60-90 mol/mol%.
  • other phospholipids may be contained in the phospholipid bilayer, such as phospholipids having a Tm value not higher than body temperature, such as dimyristate lecithin (DMPC).
  • DMPC dimyristate lecithin
  • Such a phospholipid may be present in the liposome in any suitable amount as long as it does not significantly lower the phase transition temperature of the liposome preparation to below body temperature. The appropriate amount can be easily determined by a person of ordinary skill in accordance with conventional techniques.
  • cholesterol may also be included in the liposome preparation of the present invention.
  • Cholesterol has a function of regulating membrane fluidity, and when 50% (mol/mol) of cholesterol is added to the liposome membrane, the phase transition of the liposome membrane disappears. Papahadjopoulos and other cholesterol is called "Fluidity buffer". Because cholesterol is added to phospholipids below the phase transition temperature, the membrane can be reduced in order to increase membrane fluidity. When the temperature is higher than the phase transition temperature, cholesterol is added. This can increase the orderly alignment of the film and reduce the fluidity of the film.
  • the content of cholesterol may be 2-60 mol/mol%, 5-55 mol/mol% or 10 - 50 mol/mol% of the total moles of each component of the liposome. More specifically, the content of cholesterol may be 15-45 mol/mol%, for example, 20-40 mol/mol%, based on the total moles of each component of the liposome.
  • the amount of cholesterol which can be contained in the liposome of the present invention can be determined by a person of ordinary skill by routine experimentation.
  • the phospholipid bilayer may also contain other excipients, particularly excipients which can further modify the surface characteristics of the liposome, thereby Liposomes have better in vivo behavior.
  • excipients include, for example, lipid materials modified with a hydrophilic polymer, examples of which are, for example, polyethylene glycol-modified distearoylphosphatidylethanolamine
  • the excipient may also be a membrane or the like modified with a specific antibody or ligand.
  • the molar ratio may be 0.1-20 mol/mol%, preferably 0.3-18 mol/mol%, more preferably 0.5- of the phospholipid. 15 mol/mol%, especially 0.8-12 mol/mol%, such as 1-10 mol/mol%, or 2-8 mol/mol%, 2.5-7 mol/mol%, 3-6 mol/mol%, etc. .
  • the molecular weight of the polyethylene glycol may be, for example, 400 to 20,000 Dalton, preferably 600 to 15,000 Dalton, more preferably 800 to 10,000 Dalton, especially 1000 to 8000 Dalton.
  • polyethylene glycol suitable for use in the present invention can also be readily determined by one of ordinary skill in the art based on routine experimentation.
  • the liposomal formulation of the invention is a small single compartment liposome and should have a suitable particle size.
  • the pharmaceutical preparation has a particle size of from 30 to 80 nm, more preferably from 35 to 70 nm, particularly preferably from 40 to 60 nm.
  • the particle size of the liposome can be determined by means of a particle size analyzer or an electron microscope.
  • the liposome particles may not have a completely uniform particle size, but may span a range of particle sizes. Therefore, in the liposome pharmaceutical preparation of the present invention, the presence of liposome particles outside the specified particle size range is not excluded as long as it does not significantly affect the properties of the liposome preparation or pharmaceutical preparation of the present invention. .
  • the liposome of the present invention can be prepared by various suitable methods including, for example, a film dispersion method, an injection method, an ultrasonic dispersion method, a freeze drying method, a freeze-thaw method, and the like.
  • a film dispersion method an injection method, an ultrasonic dispersion method, a freeze drying method, a freeze-thaw method, and the like.
  • the starting system when preparing liposomes it can be divided into: (1) preparation method based on dry lipid film and lipid powder, (2) emulsion-based method, and (3) mixed stock Group-based liposome preparation technology, (4) liposome preparation method based on a three-phase mixture of ethanol, phospholipid and water.
  • the encapsulation of the drug can be achieved by passive drug loading or active drug loading. This These methods are available in a number of liposome review articles.
  • the drug may be encapsulated in a liposome by a variety of suitable methods to prepare a liposome pharmaceutical preparation including, for example, an active drug loading method and a passive drug loading method.
  • Active drug loading is usually carried out by a gradient method, such as ammonium sulfate gradient method, that is, using an ammonium sulfate solution as an aqueous phase, first preparing liposomes containing both ammonium sulfate and the like, and then removing the ammonium sulfate in the aqueous solution of the lipid to make the lipid
  • the concentration of ammonium sulfate in the plastid forms a concentration gradient with ammonium sulfate in the outer aqueous phase, and the H4+ of the internal aqueous phase decomposes into H3 and H+, resulting in a difference in the concentration of H+ ions in the body and outside the liposome, ie, the pH gradient
  • the pharmaceutically active ingredient in the liposome pharmaceutical preparation of the present invention is a multivalent ion drug.
  • multivalent ion drug refers to a drug having two or more groups that can be dissociated, the dissociation group having a dissociation constant pKa between 4.5 and 9.5, thereby allowing the drug to be pKa There are multiple positive charges or multiple negative charges in the range of values.
  • the dissociation constant pKa is between 5.0 and 9.5. More preferably, the dissociation constant pKa is between 5.5 and 9.5. Particularly preferably, the dissociation constant pKa is between 6.0 and 9.0, especially between 6.5 and 9.0.
  • One of ordinary skill can readily determine the pKa value of each detachable group of the ionic drug according to conventional techniques.
  • the multivalent ion drug may include, but is not limited to, an anticancer drug, for example, for lung cancer (such as non-small cell lung cancer), pancreatic cancer, breast cancer, rectal cancer or multiple myeloma, liver cancer, cervix
  • the multivalent ion drug is mitoxantrone, vincristine, vinorelbine or vinblastine. More preferably, the multivalent ion drug is mitoxantrone, and optionally in combination with at least one other drug, such as an antineoplastic agent such as vincristine, vinorelbine or vinblastine. Wait.
  • the multivalent ion drug may also be the above drug. Any one or a combination of two or more, such as a combination of two anticancer drugs, a combination of one or more anticancer drugs with other drugs such as immunopotentiators, and two or more other drugs Combination, etc.
  • the liposome drug of the present invention may optionally include other one or more non-polyvalent ion drugs in addition to the above multivalent ion drug, and the non-polyvalent ion drug may be It is administered in combination with the above multivalent ion drug.
  • Such combined administration includes administration of the respective combinations in one formulation, and also in combination in the form of separate unit dosage forms.
  • the drug as an active ingredient mentioned in the present invention includes not only its prototype form but also derivatives thereof such as solvates (such as hydrates and alcohol adducts), prodrugs and other physiologically acceptable substances.
  • solvates such as hydrates and alcohol adducts
  • prodrugs and other physiologically acceptable substances.
  • Derivatives, prodrugs and other physiologically acceptable derivatives of pharmaceuticals as well as active metabolites and the like are well known to those of ordinary skill in the art.
  • the liposome pharmaceutical preparation of the present invention may further comprise a polyvalent counterion having two or more charges opposite to the active ingredient.
  • the polyvalent counterion include, but are not limited to, organic acid radicals such as the following acid salts of saturated or unsaturated organic acids: citric acid, tartaric acid, fumaric acid, oxalic acid, malonic acid, succinic acid, malic acid, and horses. Acid, etc., inorganic acid radicals such as sulfate, phosphate, and the like. Among them, citrate, sulfate or phosphate is preferred.
  • the polyvalent counterion may also be an amino acid such as cystine or the like.
  • the multivalent counterion can form an insoluble precipitate with a drug of interest encapsulated in the liposome, such as a multivalent ion drug, thereby stabilizing the presence of the multivalent ion drug in the liposome.
  • the liposome pharmaceutical preparation of the present invention may optionally further comprise other excipients and carriers well known in the pharmaceutical art, such as sucrose, histidine, antioxidants, stabilizers, dispersants, preservatives, dilutions. Agents, buffer substances, solvents, salts that change the osmotic pressure, and the like.
  • the invention provides a method of preparing a liposome pharmaceutical formulation of the invention, comprising first preparing a liposome formulation of the invention described above, and subsequently administering a pharmaceutical composition of interest to the liposome formulation at a suitable Incubate together under conditions.
  • the method for preparing a liposome pharmaceutical preparation of the present invention comprises the following steps: (1) preparing a liposome suitable The lipid adjuvant is dissolved in a suitable organic solvent such as t-butanol or cyclohexane, and lyophilized to obtain a lyophilized powder; (2) lyophilized the lyophilized powder using a solution containing a counterion of the active ingredient of the objective drug to form a blank Liposomes; (3) using a suitable method, such as dialysis, or column chromatography to remove the counterion of the liposome in vitro, thereby forming a counter ion gradient inside and outside the lipid membrane; and (4) incubating the drug and liposome , get liposome drugs.
  • a suitable organic solvent such as t-butanol or cyclohexane
  • the lipid is a phospholipid, especially a phospholipid having a higher phase transition temperature, such as phosphatidylcholine, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, bis-lipoic lecithin (DPPC) or bis-stearic acid Lecithin (DSPC) or any combination thereof.
  • the lipid further contains cholesterol in a ratio of, for example, 2 to 60 mol/mol%, 5 to 55 mol/mol% or 10 to 50 mol/mol%. More specifically, the content of cholesterol may be 15-45 mol/mol%, for example, 20-40 mol/mol%, based on the total moles of each component of the liposome.
  • the person skilled in the art can determine the specific content of cholesterol according to the specific requirements of the liposome transformation temperature to be obtained and the desired properties, which are within the capabilities of the skilled person.
  • the encapsulation efficiency of the drug in the liposome can be determined by conventional techniques.
  • the method for measuring the encapsulation efficiency of the liposome includes an ultrafiltration method, a dialysis method, a column stratification method, a microcolumn centrifugation method, and the like.
  • the ultrafiltration method has high requirements on experimental equipment, so it is not used; column chromatography has a low drug content due to dilution of a large amount of eluent, which makes the determination of the content difficult, and a large amount of dilution also causes drug leakage in the liposome; It can be seen from the obtained test data that the encapsulation efficiency of the dialysis method is low, which may cause the rupture of the liposome after dilution, and the dialysis takes a long time, so it is not applicable; the encapsulation efficiency is determined by the microcolumn centrifugation method, and the time is short.
  • the liposome solution has a small dilution factor and does not require expensive equipment.
  • the liposome drug of the invention not only ensures effective encapsulation efficiency and sufficient drug loading, but also ensures that the drug is not released from the liposome during the in vitro storage period, and the liposome drug is also ensured. During the blood circulation, significant leakage does not occur and toxicity is increased.
  • An important significant effect of the liposome drug of the present invention is that it effectively accelerates the release rate of the drug, improves the therapeutic index of the liposome, and has a significantly longer half-life than the free drug, and is significantly more toxic than the existing product. Reduced, effective use of drugs Therapeutic effect.
  • a liposome pharmaceutical preparation made of a phospholipid such as hydrogenated soybean lecithin (HSPC) or bis-lipoic acid lecithin (DPPC) has a markedly reduced toxicity and a marked improvement in the therapeutic index.
  • the phospholipid bilayer consists of bis-myristate lecithin (DMPC) the release rate of the drug is released too quickly, resulting in greater toxicity and even less safety than unencapsulated drugs.
  • the small single-chamber liposome preparation of the present invention can accelerate the rate of drug release because if the drug-to-lipid ratio is fixed, it is smaller than the large single-chamber liposome preparation.
  • the single-chamber liposome formulation will contain more liposome particles, each containing a small size drug precipitate inside. Smaller drug precipitates have a larger specific surface area, so under the same conditions, there will be a faster dissolution rate.
  • the liposome pharmaceutical preparations of the invention should be made of a suitable phospholipid to achieve efficient release of the encapsulated drug within the target tissue, particularly the tumor.
  • the phospholipid bilayer of the liposome pharmaceutical preparation of the present invention consists of a phospholipid having a higher phase transition temperature.
  • HSPC hydrogenated soybean lecithin
  • DPPC bisphosphonate lecithin
  • the toxicity of the prescription is significantly reduced, and the therapeutic index is significantly improved.
  • the phospholipid bilayer consists of dimyristate lecithin (DMPC), the release rate of the drug will be released too quickly, resulting in greater toxicity and even less safety than unencapsulated drugs.
  • the liposome drug of the present invention can be administered to a patient in need of such treatment by a conventional administration route in the art.
  • the liposome drug is formulated for parenteral administration.
  • the liposome drug is administered by injection.
  • the invention also provides a method of treating a disease, particularly a tumor, in a patient, the method comprising administering to a patient in need of such treatment a liposomal pharmaceutical formulation of the invention.
  • heat therapy such as radiothermal therapy
  • the patient is a mammal, preferably a human.
  • the present invention also relates to the use of the above liposome preparation or liposome pharmaceutical preparation for the preparation of a medicament for treating a tumor patient.
  • the invention is further illustrated by the following examples. These examples are for illustrative purposes only, and they should not be construed as limiting the invention. Part I: Preparation of liposomes
  • Phospholipids such as hydrogenated soy lecithin (HSPC) (or double palmitate lecithin (DPPC) or dimyristyl lecithin (DMPC)), cholesterol (Choi) according to (10: 1 to 1:1) moles
  • HSPC hydrogenated soy lecithin
  • DPPC double palmitate lecithin
  • DMPC dimyristyl lecithin
  • Choi cholesterol
  • the lyophilized powder is hydrated at 60-65 with a (50-1000 mM) ammonium sulfate solution, a citric acid solution or a transition metal metal sulfate solution (such as a nickel sulfate solution), and then shaken for about 1 hour to obtain unevenness.
  • Multi-chamber liposomes The particle size of the liposome is then reduced using a microfluidic device or a high pressure extrusion device.
  • the obtained sample was diluted 200-fold with a 0.9% NaCl solution, and then detected by NanoZS.
  • the buffer of the blank liposome external phase was then removed using an ultrafiltration unit to create a transmembrane dynamic gradient.
  • the mitoxantrone hydrochloride solution (10 mg/mL) was added to the blank liposome at a suitable ratio of lipids, and the drug was loaded at 60-65. After about 1 h of incubation, the encapsulation efficiency was determined using gel exclusion chromatography.
  • Phospholipids such as hydrogenated soy lecithin (HSPC) (or double palmitate lecithin (DPPC) or dimyristyl lecithin (DMPC)), cholesterol (Choi) according to (10: 1 to 1:1) moles
  • HSPC hydrogenated soy lecithin
  • DPPC double palmitate lecithin
  • DMPC dimyristyl lecithin
  • Choi cholesterol
  • the mixture is mixed with distearylphosphatidylethanolamine (DSPE-PEG) modified with polyethylene glycol at a molar ratio of 0.1% to 20% of the phospholipid.
  • DSPE-PEG distearylphosphatidylethanolamine
  • the above mixture is dissolved in an organic solvent such as t-butanol or cyclohexane to give a clear solution. It is then freeze dried using conventional techniques to give a uniform lyophilized powder.
  • the lyophilized powder is hydrated with a (50-1000 mM) ammonium sulfate solution, a citric acid solution or a transition metal metal sulfate solution (such as a nickel sulfate solution), and then vortexed for about 1 hour to obtain an uneven multi-chamber fat. Platinum.
  • the particle size of the liposomes is then reduced using a microfluidic device or a high pressure extrusion device. Will get The sample was diluted 200-fold with a 0.9% NaCl solution and tested with NanoZS.
  • the buffer of the blank lipid external phase is then removed using an ultrafiltration device to form a transmembrane dynamic gradient.
  • the mitoxantrone hydrochloride solution (10 mg/mL) was added to the blank liposome according to the appropriate ratio of lipids, and the drug was loaded at 60-65. After about 1 h of incubation, the encapsulation efficiency was determined using gel exclusion chromatography.
  • HSPC, Choi and DSPE-PEG2000 were mixed in a mass ratio of (3:1:1) and dissolved in 95% t-butanol to obtain a clear solution. After lyophilization, a uniform lyophilized powder was obtained. The lyophilized powder was hydrated with 300 mM ammonium sulphate, and then shaken for 1 h to obtain heterogeneous multi-chamber liposomes. The final phospholipid concentration was 96 mg/mL. The microcapsule device is then used to reduce the particle size of the liposomes.
  • the obtained sample was diluted 200-fold with a 0.9% NaCl solution, and then detected by NanoZS, the average particle size of the particles was about 60 nm, and the main peak was concentrated between 40-60 nm. Thereafter, the ammonium sulfate in vitro phase of the blank lipid was removed using an ultrafiltration apparatus, and the external phase was replaced with 250 mM sucrose and 50 mM glycine to form a transmembrane ammonium sulfate gradient. The mitoxantrone hydrochloride solution (10 mg/mL) was added to the blank liposome at a ratio of the lipid to 16:1, and the drug was loaded at 60-65. After about 1 h of incubation, gel exclusion chromatography was used to demonstrate an encapsulation efficiency of approximately 100%. This prescription was named plm 60» Example 3
  • HSPC, Choi and DSPE-PEG2000 were mixed at a mass ratio of 3:1:1, and dissolved in 95% t-butanol to obtain a clear solution.
  • a homogeneous lyophilized powder is obtained.
  • the lyophilized powder was hydrated with a 300 mM ammonium sulfate solution at 60-65, followed by shaking for 1 h to obtain a heterogeneous multi-chamber liposome.
  • the final phospholipid concentration was 96 mg/mL.
  • the particle size of the liposomes is then reduced using high pressure extrusion. Will get The sample was diluted 200-fold with NaCl solution and detected by NanoZS. The average particle size of the particles was about 85 nm.
  • the ammonium sulfate in vitro phase of the blank lipid was then removed using an ultrafiltration apparatus, and the external phase was replaced with 250 mM sucrose and 50 mM glycine to form a transmembrane ammonium sulfate gradient.
  • the mitoxantrone hydrochloride solution (10 mg/mL) was added to the blank liposome at a ratio of the ratio of the lipid to the ratio of 16:1, and the drug was loaded at 60-65. After about 1 h of incubation, gel exclusion chromatography was used to demonstrate an encapsulation efficiency of about 100%. This prescription is named plm 85.
  • the mitoxantrone liposome plmlOOo was prepared in the same manner as in Example 3, and its formulation was the same as that of plm60 and plm85, but the particle size of the liposome was 100 nm.
  • Example 5 The mitoxantrone liposome plmlOOo was prepared in the same manner as in Example 3, and its formulation was the same as that of plm60 and plm85, but the particle size of the liposome was 100 nm.
  • Example 7 Mix DMPC, Choi and DSPE-PEG2000 in a mass ratio of 3:1:1, and the other operations are the same as in Example 2. This prescription is named plm60-dmpc.
  • Example 7
  • mitoxantrone and acesulfame were drug-loaded using a pH gradient. If a certain concentration of ammonium chloride is added to the release medium, the free ammonia molecules will diffuse into the inner phase space of the liposome by means of a gradient, thereby increasing the pH of the internal phase and protonating the drug of the internal phase. A neutral drug that can diffuse across the membrane. This process also accelerates the dissolution of the liposomes in the body. The rate at which the drug is released from the liposome is controlled by both precipitation and membrane permeability. The conditions of release were as follows, and the liposomes were diluted 25-fold with a release medium.
  • the release medium contained 2, 10 and 40 mM ammonium chloride, isotonic, pH 7.4.
  • the diluted liposomes were placed in a dialysis bag, and 2 mL of the liposome dilution was dialyzed against a 400 mL of release medium for release. Samples were taken at different time points for analysis and continued for 96 hours. The obtained data is returned.
  • the half-life of drug release in pld60 in release media containing 2, 10 and 40 mM ammonium chloride was 94.3, 31.9 and 11.2 hours, respectively. For plm60, there was no significant release in the three release media. Since there is no difference in liposome composition and particle size between the two formulations, the difference in drug release kinetics may be due to differences in drug properties.
  • mitoxantrone contains two physiological pHs. Dissociated group.
  • This embodiment fully demonstrates that a drug containing multiple dissociation groups such as mitoxantrone, when using active drug-loading technology, forms a more complex deposition structure with the counterion, thereby significantly delaying the release of the drug in the in vitro release medium. freed.
  • drugs with a single dissociation group such as Avastriol, the use of a small particle size prescription may result in a drug that is released too quickly and will be released faster in a release medium that does not contain plasma.
  • Release conditions 1 The liposomes were diluted 25-fold with the release medium.
  • the release medium contained 50% human plasma, wasotonic with glucose, pH 7.4, and the others were the same as in Example 9. Regression of the obtained data indicated that the release half-life of plm60 was 56.4 hours, whereas plm85 had no significant release under the above conditions.
  • Release conditions 2 The release medium contains 50% human plasma and 20 mM ammonium chloride.
  • the other operation method is the same as that of the embodiment 9. Regression of the obtained data revealed that the release half-life of plm60 was 26.2 hours, while the release half-life of plm85 was 36.7 hours.
  • the release half-life of plm60-DMPC-0.1 was 15 hours. This experiment shows that switching to a low Tm phospholipid can also speed up drug release. However, the increased transition of the drug's release behavior can lead to excessive toxicity of the drug in the body, which can also be found in subsequent experiments.
  • mice used in the experiment were about 20 g of Kunming male mice.
  • S-180 sarcoma cells were inoculated into the right side of the mouse in a ratio of 5 X 10 5 .
  • the drug is administered intravenously, and the mice are sacrificed at a certain time point after administration, and samples of various tissues and organs are taken, and the content of mitoxantrone is determined.
  • the tissues measured include heart, liver, spleen, lung, kidney, intestine, marrow, and tumor.
  • the experimental results show that PLM60 has a very obvious targeting effect on tumor tissues. Table 2 and Figure 2 give more detailed experimental data.
  • the experimental animals used were the same as in Example 12, and the three prescriptions were administered at a dose of 4 mg/kg, which was intravenously injected into mice. Comparing the data in Figures 3 and 3, it can be found that: When the composition of the lipid bilayer is significantly changed, the pharmacokinetic behavior of the liposome drug in vivo is significantly changed.
  • the MRT values for the three formulations of PLM60-DPPC, PLM60-DMPC-0.1 and PLM60-DMPC were 14.22, 7.914 and 10.123 hours, respectively.
  • the difference in prescription between PLM60-DPPC and PLM60-DMPC is that the phospholipids used have different lengths of hydrocarbon chains of 16 and 14 carbons, respectively. The difference in length of the acyl chain can significantly affect the permeability of the phospholipid bilayer.
  • the DPPC has a Tm value of 41 degrees and the DMPC has a Tm value of 23 degrees.
  • PLM60-DMPC-0.1 The difference between PLM60-DMPC-0.1 and PLM60-DMPC is that the degree of PEGylation is different.
  • the release of liposomes in plasma is related to two factors, one is the release of the drug across the phospholipid bilayer, and the other is related to the phagocytosis of lipoproteins and the reticuloendothelial system. Since the PEGylation of PLM60-DMPC-0.1 liposomes is incomplete, it is greatly affected by the release caused by plasma components.
  • Table 3 Comparison of pharmacokinetic parameters of different prescriptions in mice
  • mice were used, all male, weighing 18-22 grams. A total of 9 groups, 10 in each group. The first group was given free mitoxantrone at a dose of 6 mg/kg. The remaining 8 groups were given PLM60, PLM60-DPPC, PLM60-DMPC-0.1 and PLM60-DMPC doses of 6 and 12 mg/kg, respectively. The changes in body weight of the mice were observed and the time of death of the mice was recorded. After the mice died, they were dissected and autopsied. See Table 5 for the deaths of the free drug group and the liposome group.
  • mice Male C57 mice weighing 18 to 22 grams were used to compare the toxicity of plm60, plm85 and plmlOO. At a dose of 9 mg/kg, it was found that under the above conditions, the three prescriptions resulted in a change in body weight of the mice, demonstrating that there was no significant difference in toxicity between the three sites under the above test conditions. The same dose of the free drug group lost more than 30% of the body weight, and 20% of the mice died. The fifth part of the anti-tumor effect in vivo
  • the ascites tumor-bearing mice inoculated with S180 tumor cells for 7 days were sacrificed by cervical dislocation, milky white viscous ascites was drawn, diluted with RPMI 1640 medium, and the number of tumor cells was adjusted after dilution. It is 2.5 x 10 6 cells/ml.
  • the tumor cell suspension was inoculated into the subcutaneous subcutaneous tissue of the forearm of KM male mice weighing 18 ⁇ 22 g, the inoculation volume was 0.2 ml, and the tumor-containing cells were about 5 x l0 5 . After the inoculation, the remaining tumor cell suspension was in the light. Microscopically counted, the number of viable cells was > 95%. A total of 80 mice were inoculated.
  • mice with clear tumor boundaries and a tumor diameter of about 5 were selected, and the animals were stratified according to the size of the tumor, and then randomly divided into groups according to body weight. There were 7 blank control groups, 4 PLM60 4, 6 mg/k group, mitoxantrone free drug 4, and 6 mg/kg group. Intravenous administration was performed in groups.
  • Mice were sacrificed by cervical dislocation on the 21st day after inoculation, and the tumor pieces were dissected and weighed.
  • the experimental results were analyzed by t test.
  • Table 6 shows that the mitoxantrone liposomal (PLM60) 4, 6 mg/k dose group significantly inhibited the growth of S180 solid tumors.
  • B12 mice with L1210 ascites tumors inoculated for 7 days were sacrificed by cervical dislocation. Milky white viscous ascites was aseptically diluted and diluted in RPMI 1640 medium. After dilution, the number of tumor cells was adjusted to 2.5 X 10 5 cells/ml.
  • the tumor cell suspension was intravenously inoculated into 7-8 weeks old BDF1 male mice, the inoculation volume was 0.2 ml, and the tumor-containing cells were about 5.0 x 10 4 . After the inoculation was completed, the remaining tumor cell suspension was counted under a light microscope. The number of viable cells was > 95%schreib A total of 62 mice were inoculated.
  • the drug was administered in groups. After the administration, the animals were normally reared, the rats were weighed three times a week, and the deaths of the animals were observed and recorded every day, and the number of days of survival was calculated. The experiment was observed up to 60 days after inoculation.
  • mice in the blank group died on the 11th-14th day after inoculation; all the animals in the three dose groups of the mitoxantrone free drug died during the 11-17th day after the inoculation; All animals in the PLM60 2 mg/k dose group died during the 15th to 29th day after vaccination. In the 6 mg/k dose group, only one animal died on the 39th day after vaccination, and the 8 mg/k dose group had no animal death.
  • N a All animals did not die at the end of the 60-day observation period and the survival time could not be calculated.
  • the formulation of the different granules of mitoxantrone liposome for the therapeutic effect of L1210 ascites tumor and the mode of data processing were the same as those of Example 19.
  • a total of 5 experimental groups were set up, the blank group, the free drug group, the plm60, plm85 and plmlOO groups.
  • the dose administered per group was 4 mg/kg.
  • the experimental results show that the small particle size liposome has a better therapeutic effect.
  • the experimental results are shown in Table 9. Effect of L1210 ascites tumor on survival time of BDF1 mice
  • N b Only a few animals died at the end of the 60-day observation period, and the median survival time could not be calculated.

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Description

一种脂质体药物制剂及其制备方法
技术领域 本发明涉及一种脂质体制剂以及其中包裹了药物的脂质体药物 制剂，特别是涉及米托蒽醌的脂质体药物制剂。本发明还涉及所述脂 质体、 脂质体药物制剂的制备方法和用途。
技术背景 脂质体可以作为许多药物的载体，特别是作为抗肿瘤药物（尤其 是化疗药物）的载体， 它可以减少药物在正常组织的分布， 增加药物 在肿瘤组织的蓄积量，从而改善药物的治疗指数。脂质体之所以可以 被动的靶向肿瘤， 跟肿瘤的生理特点有关。 肿瘤由于快速生长， 其血 管间隙可以达到 100-780nm， 正常血管内皮细胞之间的间隙通常在 2nm 左右。 因此， 如果脂质体可以在血液中较长时间的循环， 并且 脂质体的粒度 <200nm，它就可以被动的聚集在肿瘤区。 这是因为： 静 脉注射给药后， 小粒度的脂质体在血液循环的过程中，无法进入正常 组织， 但是却可以有效的穿透肿瘤区的血管， 到达治疗部位。
但是，要想真正的实现脂质体的治疗优势是不容易的，必须满足 以下四点要求： 1)药物能够以有效的包封效率和足够的载药量包封在 脂质体内； 2 )在体外贮存期内， 药物不会从脂质体中释放出来； 3 ) 脂质体药物在血液循环的过程中， 药物不会发生显著的渗漏； 和 4 ) 当脂质体聚集在肿瘤区以后， 药物能够有效地释放，从而发挥药物的 治疗作用。 就目前的脂质体技术看， 已经较好地解决了前三个问题， 而使脂质体药物在体内合理释放成为了研究重点和难点。如何有效的 控制某些脂质体药物在靶向肿瘤区后合理的释药，是开发脂质体药物 时必须解决的一个技术问题。这对于某些药物尤为重要，如米托蒽醌。 加拿大的脂质体研究小组， 使用氢化大豆卵磷脂（HSPC )和胆 固醇作为磷脂双层， 300mM的柠檬酸梯度进行载药， 脂质体的粒度 在 lOOnm左右， 结果发现制备得到的脂质体的抗肿瘤效应不如游离 的米托蒽醌。为了改变脂质体的治疗效果，最终他们把磷脂双层的组 成改为双肉豆寇酸卵磷脂（DMPC )和胆固醇， 得到了治疗指数改善 的处方。但是由于 DMPC的相转变温度在 21度左右，在储存期内药 物的渗漏有可能增加， 处方会不稳定 ( Liposomal formulations of mitoxantrone, US5,858,397 )»
美国的 Neopharm公司则使用另外一种技术开发米托蒽醌脂质 体。他们在磷脂的双分子层中添加荷负电的心磷脂。 由于心磷脂可以 和米托蒽醌强烈相互作用，因此米托蒽醌可以通过被动载药的方式被 插入磷脂双分子层中。此种技术和主动载药技术有差别。使用主动载 药技术， 药物将以沉淀的形式沉积在脂质体的内水相中。该公司的产 品经过一期的临床研究发现。脂质体药物和游离药物相比，可以增加 偶发性感染的几率。 出于安全性的考虑， 该产品被停止研发（米托蒽 醌的脂质体制剂， CN01817424.8 )。
中国的常州太平洋药物研究所也就米托蒽醌脂质体申请了一项 专利 （米托蒽醌或盐酸米托蒽醌脂质体注射剂及其制备工艺， CN200410041612.1 ) 。 该专利申请中使用了传统的 pH值梯度法进 行载药。 申请保护的是一个特定比例的处方。在该专利申请中， 并没 有考虑到磷脂组成、 内水相緩冲盐种类、脂质体粒度、 药脂比等因素 对脂质体的疗效和毒性的影响。
四川大学华西药学院的张志荣等人也曾研究过米托蒽醌脂质体 制剂。 他们使用的磷脂为相转变温度为 0度左右的大豆磷脂酰胆碱 (其商品名为 EPIKURON 200 )， 粒度约为 60nm左右。 此研究学者 只进行了药代动力学的研究，没有进一步研究所获得的脂质体制剂的 毒性和疗效。有关内容考参考米托蒽醌长循环脂质体的制备及药代动 力学的研究 ，张志荣、于波涛、段逸松，药学学报， 2002 Vol.37 No.6; 硫酸铵梯度法制备米托蒽醌长循环脂质体 ， 张志荣、 于波涛、 段逸 松、 黄园， 中国药学杂志， 2002 Vol.37 No.12; 米托蒽醌脂质体制 备工艺的研究 ， DUAN Yi-song,华西药学杂志， 2001 Vol.16 o.02„ 以上的研究中， 通常将脂质体的粒度控制在 80 ~ 150nm之间， 因为脂质体学界有一个共识， 认为 lOOnm左右的处方有最好的靶向 效率（Pharmacol. Rev. 1999 51: 691-744. )„ 可是如前所述， 仅有最 优的靶向效率是不足够的， 药物必须能够有效地从脂质体中释放出 来， 才能够发挥作用。
如上所述，在现有技术中， 为了保证药物有效的被脂质体转运到 病灶部位，要求在血液循环的过程中， 药物基本上不渗漏， 这同时却 导致了药物被靶向到肿瘤区以后，也很难从脂质体中释放出来。在常 规的脂质体制备工艺中，通常使用主动载药技术来包裹目的药物，其 中脂质体包封的药物是以胶态沉淀的方式存在的， 不具有生物活性。 只有药物有效的从脂质体释放出来，才可以转变为有生物活性的治疗 性药物。 如果药物的释放过于緩慢， 即使其被有效的靶向到肿瘤区， 它也艮难起到真正的治疗作用。其治疗效果甚至还会不如未包封的药 物。
因此，现有技术中迫切需要有能够以很好靶向性递送药物、 同时 在靶组织中将其有效释放的脂质体制剂， 以及相应的脂质体药物制 剂。
发明内容 本发明意外地发现，针对某些具有多个解离基团、容易和多价反 离子形成较致密沉淀的药物， 通过将它们制成小单室的脂质体制剂 后， 可以有效地改善脂质体的治疗指数， 从而解决了上述技术问题。
因此， 本发明一方面提供了一种脂质体制剂， 其粒度为约 30-80 nm， 并且在磷脂双分子层中含有 Tm高于体温的磷脂， 从而脂质体 的相转变温度高于体温。所述磷脂的实例包括例如磷脂酰胆碱、氢化 大豆卵磷脂 （HSPC )、 氢化蛋黄卵磷脂、 双软脂酸卵磷脂（DPPC ) 或双硬脂酸卵磷脂（DSPC )或者其任何组合， 但并不局限于此。 在一个实施方案中，所述磷脂双分子层中 Tm高于体温的磷脂占 总磷脂含量的 50-100 mol/mol%，优选 55-95 mol/mol %，更优选 55-95 mol/mol %„
可选地，在本发明脂质体制剂的所述磷脂双分子层中还可以还有 其它磷脂，例如 Tm值不高于体温的磷脂，其实例有双肉豆寇酸卵磷 脂（DMPC )等。 至于这种磷脂在本发明脂质体制剂中的含量， 则是 普通技术人员可以常规确定的， 只要其不显著降低该脂质体制剂的 Tm值至低于体温即可。
在本发明的脂质体制剂中，还可以可选地含有胆固醇， 以便调节 脂质体膜的流动性。
本发明的脂质体制剂中还可以可选地含有其他的辅料，特别是可 以进一步修饰脂质体的表面特征的辅料，从而使脂质体具有更佳的体 内行为。 这样的辅料包括例如用亲水性聚合物修饰的脂类物质等。
本发明另一方面提供了一种脂质体药物制剂，其在本发明的脂质 体制剂中包含了感兴趣的药物， 尤其是多价离子药物。 因此， 本发明 涉及一种脂质体药物制剂， 其粒度在 30 ~ 80nm之间， 并且： 1 )含 有作为活性成分的多价离子药物； 2 )在磷脂双分子层中含有 Tm高 于体温的磷脂， 从而脂质体的相转变温度高于体温； 并且可选地 3 ) 含有其它药物和 /或其他脂质体药物制剂可接受的辅料。 优选地， 所 述脂质体药物制剂粒度的主峰集中在 35-75 nm左右， 尤其是 40 ~ 60nm左右。
本发明又一方面提供了上述脂质体药物制剂的制备方法，其中包 括以下步骤： （1) 利用 Tm值高于体温的磷脂以及可选地其它磷脂和 /或胆固醇， 制备脂质体； 和 (2)将感兴趣的药物、 特别是多价离子药 物包封在脂质体内。
本发明还提供了一种疾病治疗方法，包括对有需要的对象施用本 发明的脂质体药物制剂。 优选地， 所述对象是哺乳动物， 尤其是人。 附图说明
附图 1: PLM 60在昆明小鼠体内的药代学行为及其与游离米托 蒽醌药代对比。其中 PLM表示 PEG化的米托蒽醌脂质体， FM为米 托蒽醌游离药物，横坐标为时间， 单位为小时。纵坐标为米托蒽醌的 血浆浓度， 单位为 ug米托蒽醌 /mL血浆。
附图 2: PLM 60和 FM在小鼠肿瘤内分布图。 其中 PLM 60为 PEG化的米托蒽醌脂质体， FM为米托蒽醌游离药物， 横坐标为时 间， 单位为小时。纵坐标为米托蒽醌的在肿瘤组织中的浓度， 单位为 u 米托蒽醌 /g肿瘤组织。
附图 3: 不同处方在小鼠体内的药代学对比。 横坐标为时间， 单 位为小时， 纵坐标为米托蒽醌的血浆浓度， 单位为 米托蒽醌 /mL 血浆。 不同的处方给药剂量均为 4 mg/kg。 具体实施方式
脂质体是由磷脂、胆固醇等为膜材包合而成。这两种成分不但是 形成脂质体双分子层的基础物质，而且本身也具有极为重要的生理功 能。
脂质体膜的物理性质与介质温度有密切关系。当升高温度时脂质 双分子层中酰基侧链从有序排列变为无序排列，这种变化引起脂膜的 物理性质一系列变化，可由 "胶晶 "态变为"液晶"态，膜的横切面增加， 双分子层厚度减小，膜流动性增加，这种转变时的温度称为相变温度 (phase transition temperature)。 可借助差示扫描量热法 (Differential Scanning Calorimertry)， 电子自旋共振光 i普 (Electron Spins Resonance简称 ESR)等测量脂膜的相变温度， 脂质体膜的相变温度 取决于其组成磷脂的种类，一般而言，酰基侧链越长则相变温度越高， 反之链短则相变温度亦低，如二肉豆蔻碑脂酰胆碱相变温度为 24°C， 而二棕榈酸磷脂酰胆碱及二硬脂酰磷脂酰胆^则分别为 41°C和 58°C。膜的流动性是脂质体的一个重要物理性质，在相变温度时膜的 流动性增加，被包裹在脂质体内的药物具有最大释放速率， 因而膜的 流动性直接影响脂质体的稳定性。
在一个实施方案中，本发明提供了一种脂质体制剂，其粒度为约
30-80 nm, 并且在磷脂双分子层中含有 Tm高于体温的磷脂，从而脂 质体的相转变温度高于体温。
优选地，本发明的脂质体药物制剂由相转变温度 Tm较高的磷脂 制成 > 例如磷脂酰胆碱。其中如果磷脂酰胆碱的 Tm高于体温， 则其 烃链的长度优选不少于 16个碳。 优选本发明的磷脂包括但不限于氢 化大豆卵磷脂、 氢化蛋黄卵磷脂、 双软脂酸卵磷脂（DPPC )或双硬 脂酸卵磷脂（DSPC )或者其任何组合。
在本发明的脂质体制剂中，其中所述磷脂双分子层中 Tm高于体 温的磷脂占总磷脂含量的约 50-100 mol/mol%，优选约 55-95 mol/mol %， 更优选约 60-90 mol/mol %。 可选的， 在所述磷脂双分子层中可 含有其它磷脂，例如 Tm值不高于体温的磷脂，如双肉豆蔻酸卵磷脂 ( DMPC )等。 这种磷脂可以任何合适的量在脂质体中存在， 只要其 不显著地将脂质体制剂的相变温度降低到低于体温即可。普通技术人 员根据常规技术可以很容易地确定该合适用量。
优选地，在本发明的脂质体制剂中还可含有胆固醇。胆固醇具有 调节膜流动性的作用，当在脂质体膜中加入 50%(mol/mol)胆固醇时， 可使脂质体膜相变消失。 Papahadjopoulos 等称胆固醇为 "流动性緩 冲剂 "(Fluidity buffer), 因在低于相变温度时磷脂中加胆固醇则可使 膜减少有序排列而增加膜流动性，高于相变温度时加胆固醇则可增加 膜的有序排列而减少膜的流动性。在本发明的脂质体制剂中，胆固醇 的含量可以是脂质体各成分总摩尔数的 2-60 mol/mol%、 5-55 mol/mol%或者 10 - 50 mol/mol%。 更具体地， 胆固醇的含量可以是 脂质体各成分总摩尔数的 15-45 mol/mol%， 例如 20-40 mol/mol%。 普通技术人员可以通过常规实验确定在本发明的脂质体中可以包含 的胆固醇含量。
应当明了，在本发明的脂质体中，磷脂双分子层中也可以含有其 他的辅料，特别是可以进一步修饰脂质体的表面特征的辅料，从而使 脂质体具有更佳的体内行为。这样的辅料包括例如用亲水性聚合物修 饰的脂类物质，其实例例如有聚乙二醇修饰的二硬脂酰磷脂酰乙醇胺
(DSPE-PEG)， 聚乙二醇修饰的二硬脂酰磷脂酰甘油 (DSPG-PEG)， 聚乙二醇修饰的胆固醇 (chol-PEG)，聚维酮修饰的二硬脂酰磷脂酰乙 醇胺（DSPE-PVP)， 聚乙二醇修饰的二硬脂酰磷脂酰甘油 (DSPG-PVP), 或者聚乙二醇修饰的胆固醇 (chol-PVP)。 所述辅料还 可以是用特定抗体或配基修饰的膜材等。普通技术人员可以根据常规 技术确定这样的辅料在本发明脂质体中的用量，例如其摩尔比可以是 磷脂的 0.1-20 mol/mol % , 优选 0.3-18 mol/mol% , 更优选 0.5-15 mol/mol% , 尤其是 0.8-12 mol/mol%， 例如 1-10 mol/mol%， 或者 2-8 mol/mol% , 2.5-7 mol/mol%, 3-6 mol/mol%等。在采用聚乙二醇 修饰的脂类物质作为辅料的情形下， 聚乙二醇的分子量可以是例如 400-20000 Dalton,优选 600-15000 Dalton,更优选 800-10000 Dalton, 尤其是 1000-8000 Dalton, 例如 1200-5000 Dalton, 在本发明中适用 的聚乙二醇也是普通技术人员根据常规实验可以容易确定的。
本发明的脂质体制剂是小的单室脂质体， 应该具有合适的粒度。 优选地， 该药物制剂的粒度为 30 - 80nm， 更优选为 35 - 70nm， 尤 其优选 40 - 60nm。 脂质体的粒度可以通过粒度测定仪或者电子显微 镜等手段确定。在本发明中， 应当理解， 由于脂质体本身的性质以及 制备工艺的特点，脂质体颗粒不可能具有完全均一的粒度， 而是可能 跨越一个粒度范围。 因此， 在本发明的脂质体药物制剂中， 并不排除 所指明的粒度范围之外的脂质体颗粒的存在，只要其不显著影响本发 明的脂质体制剂或药物制剂的性质即可。
可以通过多种合适的方法制备本发明的脂质体，包括例如薄膜分 散法、 注入法、 超声波分散法、 冷冻干燥法、 冻融法等等。 根据制备 脂质体时的出发体系的不同， 可以分为： （1)以干燥的脂膜、脂类粉末 为基础的制备方法，（2)以乳剂为基础的方法，（3)以混合股团为基础的 脂质体制备技术， （4)以乙醇、磷脂、 水三相混合物为基础的脂质体制 备方法。药物的包封则可以以被动载药或者主动载药的方式实现。 这 些方法在很多的脂质体综述性文章中都可以得到。
在制备脂质体制剂时或之后，可以采用多种合适的方法将药物包 封在脂质体内，制成脂质体药物制剂， 包括例如主动载药法和被动载 药法。 主动载药通常采用梯度法进行， 例如硫酸铵梯度法， 即利用硫 酸铵溶液作为水相，首先制备内外均含有硫酸铵的脂质体， 然后通过 除去脂质体外水溶液中的硫酸铵，使得脂质体内的硫酸铵浓度与外水 相的硫酸铵形成浓度梯度，内水相的 H4+分解成 H3和 H+， 导致 脂质体内外的 H+离子浓度差， 即 pH梯度， 使得外水相中分子态的 药物 ¾>V内水相后遇酸性环境变成离子态，无法通过脂质双层回到外 水相， 从而使得脂质体不易泄漏，更加稳定。 被动载药可采用有机溶 剂注入法、 薄膜分散法和冻融法等进行。
在本发明中， 可以采用任何合适的药物成分。优选地， 在本发明 脂质体药物制剂中的药物活性成分是多价离子药物。术语 "多价离子 药物"指的是药物具有两个或两个以上的可以解离的基团，该可解离 基团的解离常数 pKa在 4.5 ~ 9.5之间， 从而使药物在此 pKa值范围 内带有多个正电荷或者多个负电荷。 优选地， 所述解离常数 pKa在 5.0-9.5之间。 更优选地， 所述解离常数 pKa在 5.5-9.5之间。 特别优 选地， 所述解离常数 pKa在 6.0-9.0之间， 尤其是 6.5-9.0之间。普通 技术人员可以容易地根据常规技术测定离子药物的各个可解离基团 的 pKa值。
在本发明中， 所述多价离子药物可包括、但不限于抗癌药物， 例 如用于肺癌 （如非小细胞肺癌）、 胰腺癌、 乳腺癌、 直肠癌或多发性 骨髓瘤、 肝癌、 宫颈癌、 胃癌、 前列腺癌、 肾癌和 /或膀胱癌等的预 防或治疗的抗癌药物。 因此， 在本发明的一个实施方案中， 所述多价 离子药物是多价离子抗癌药。优选地，所述多价离子药物是米托蒽醌、 长春新碱、长春瑞滨或者长春碱。 更优选地， 所述多价离子药物是米 托蒽醌，并且可选地与至少一种其它药物相组合，所述其它药物例如 可为抗肿瘤药比如长春新碱、 长春瑞滨或者长春碱等。
特别需要指出的是，在本发明中， 多价离子药物也可以是上述药 物中的任一种或两种以上的组合， 比如两种抗癌药物的组合、一种或 多种抗癌药物与其它药物比如免疫增强剂的组合、以及两种或多种其 它类药物的组合等。
另外需要说明的是，本发明中的脂质体药物除了上述的多价离子 药物以外，还可任选地同时包含其它一种或多种非多价离子药物，所 述非多价离子药物可与上述多价离子药物组合施用。所述组合施用包 括各自组合在一种制剂中施用，也包括以分开的单位剂型的形式组合 施用。
应当理解，本发明中所提及的作为活性成分的药物不仅包括其原 型形式，还可包括其衍生物，例如溶剂化物（比如水合物和醇加成物）、 前药和其它生理上可接受的衍生物、以及活性代谢物等。药物的衍生 物、前药和其它生理上可接受的衍生物以及活性代谢物等是普通技术 人员所熟知的。
本发明所述的脂质体药物制剂中还可含有具有两个或两个以上 与活性成分电荷相反的多价反离子。所述多价反离子的实例包括、但 不限于有机酸根， 比如以下饱和或不饱和有机酸的酸根： 柠檬酸、 酒 石酸、 富马酸、 草酸、 丙二酸、 琥珀酸、 苹果酸， 以及马来酸等， 无 机酸根比如硫酸根、磷酸根等。其中优选柠檬酸根、硫酸根或者磷酸 根。 此外， 所述多价反离子也可为氨基酸， 比如胱氨酸等。 不局限于 某种具体的理论，推测该多价反离子能够与脂质体内包裹的感兴趣药 物、例如多价离子药物形成难以溶解的沉淀，从而稳定多价离子药物 在脂质体内的存在。
本发明的脂质体药物制剂中还可任选地包含制药领域中所公知 的其它的赋形剂和载体， 比如蔗糖、 组氨酸、 抗氧剂、稳定剂、 分散 剂、 防腐剂、 稀释剂、 緩冲物质、 溶剂、 改变渗透压的盐等。
在一个实施方案中，本发明提供了制备本发明脂质体药物制剂的 方法， 包括首先制备本发明上述的脂质体制剂， 以及随后将感兴趣的 药物成分于该脂质体制剂在合适的条件下一起孵育。更具体而言，本 发明的脂质体药物制剂制备方法包括以下步骤： （1)将制备脂质体适 用的脂类辅料溶解于合适的有机溶剂例如叔丁醇或环己烷中，冷冻干 燥得到冻干粉末； (2)使用含有目的药物活性成分的反离子的溶液水 化冻干粉末以形成空白脂质体； （3) 采用合适的方法， 例如透析、 或 者柱层析等手段除去脂质体外的反离子，从而使脂膜内外形成反离子 梯度； 和 (4)将药物和脂质体孵育， 得到脂质体药物。 有关施用的磷 脂、 胆固醇、 辅料等的描述， 参见上文针对脂质体制剂所述。
优选地， 所述脂类是磷脂， 尤其是相变温度较高的磷脂， 例如磷 脂酰胆碱、 氢化大豆卵磷脂、 氢化蛋黄卵磷脂、 双软脂酸卵磷脂 ( DPPC )或双硬脂酸卵磷脂 （DSPC )或者其任何组合。 可选地， 所述脂类中还含有胆固醇， 其比例是例如 2-60 mol/mol%、 5-55 mol/mol%或者 10 - 50 mol/mol%。 更具体地， 胆固醇的含量可以是 脂质体各成分总摩尔数的 15-45 mol/mol% , 例如 20-40 mol/mol%。 普通技术人员可以根据待获得的脂质体相变温度的具体要求及所需 的性质而根据常规技术确定胆固醇的具体含量，这些均在技术人员的 能力范围内。
脂质体药物制剂一旦制备以后，即可通过常规技术测定药物在脂 质体中的包封率。脂质体包封率测定的方法有超滤法、透析法、柱层 析法、微柱离心法等。 超滤法对实验设备要求较高， 故未采用； 柱层 析法则由于大量洗脱液的稀释，药物含量很低，造成含量测定的困难， 且大量稀释也引起脂质体中药物渗漏；从所得试验数据可知透析法包 封率较低， 可能是稀释后引起脂质体的破裂， 且透析所需时间长， 故 不适用； 用微柱离心法进行包封率测定， 耗时短， 脂质体溶液稀释倍 数小， 且不需要昂贵的仪器设备。
本发明的脂质体药物不仅保证了有效的包封效率和足够的载药 量， 还保证了在体外贮存期内， 药物不会从脂质体中释放出来， 并且 还保证了脂质体药物在血液循环的过程中， 不会发生显著的渗漏， 而 增加毒性。本发明的脂质体药物重要的显著效果是有效的加快药物的 释放速率， 改善了脂质体的治疗指数， 并且与游离药物相比， 半衰期 明显延长， 与已有的产品相比， 毒性显著降低， 有效的发挥了药物的 治疗作用。 例如， 由氢化大豆卵磷脂 （HSPC )或双软脂酸卵磷脂 ( DPPC )这样的磷脂制成的脂质体药物制剂，处方的毒性显著降低， 并且治疗指数明显改善。相比之下，如果磷脂双层由双肉豆寇酸卵磷 脂（DMPC )组成， 药物的释放速度就会释放过快，导致较大的毒性， 甚至安全性不如未包封的药物。不局限于某种具体的理论，推测本发 明的小单室脂质体制剂可以加快药物释放速度的原因是，如果药脂比 固定，那么相比于大的单室脂质体制剂， 小的单室脂质体制剂就会含 有更多的脂质体粒子，每个脂质体内部含有小尺寸的药物沉淀。小尺 寸的药物沉淀有更大的比表面积， 因此在同样的条件下，就会有更快 的溶解速率。
此外，本发明脂质体药物制剂应该由合适的磷脂制成， 以实现所 包裹药物在靶组织、尤其是肿瘤内的有效释放。优选地， 本发明脂质 体药物制剂的磷脂双分子层由相转变温度较高的的磷脂组成。我们在 实验中发现， 如果脂质体处方使用氢化大豆卵磷脂（HSPC )或双软 脂酸卵磷脂（DPPC )这样的磷脂， 处方的毒性显著降低， 并且治疗 指数明显改善。 如果磷脂双层由双肉豆寇酸卵磷脂 （DMPC )组成， 药物的释放速度就会释放过快，导致较大的毒性，甚至安全性不如未 包封的药物。
本发明中的脂质体药物可以采取本领域常规给药途径给需要此 治疗的患者施用。在本发明的一个实施方案中，所述脂质体药物被配 制成胃肠外施用的制剂。在本发明的一个优选的实施方案中，所述脂 质体药物经注射施用。
本发明还提供了一种治疗患者中疾病、特别是肿瘤的方法，所述 方法包括给需要该治疗的患者施用本发明所述的脂质体药物制剂。优 选地， 还可以对肿瘤患者联合应用热疗法， 例如放射热疗法， 以便加 强脂质体药物制剂的治疗效果。 在本发明中， 所述患者是哺乳动物， 优选人。
本发明还涉及上述脂质体制剂或者脂质体药物制剂在制备用于 治疗肿瘤患者的药物中的用途。 下面通过实施例来进一步说明本发明。 这些实施例只是举例说 明， 它们不应当构成对本发明的限制。 第一部分： 脂质体的制备
实施例 1
脂质体制备的一般方案
1.一般方案 1
将磷脂如氢化大豆卵磷脂（ HSPC ) (或双软脂酸卵磷脂（ DPPC ) 或双肉豆寇酸卵磷脂（DMPC ) )、胆固醇 (Choi)按照（10: 1至 1: 1 ) 的摩尔比混合，溶解于有机溶剂如叔丁醇或者环己烷中，得到澄明溶 液。之后采用常规技术冷冻干燥得到均匀的冻干粉末。将此冻干粉末 用 （50-1000mM ) 的硫酸铵溶液、 柠檬酸溶液或者过渡态金属的硫 酸盐溶液（如硫酸镍溶液）等在 60-65 水化， 之后震荡约 lh， 得到 不均匀的多室脂质体。之后使用微射流设备或者高压挤出设备降低脂 质体的粒度。 将所获得的样品用浓度为 0.9%的 NaCl溶液稀释 200 倍后，用 NanoZS进行检测。之后使用超滤装置移去空白脂质体外相 的緩冲液， 以便形成跨膜动力梯度。按照合适的脂药比， 在空白脂质 体中加入米托蒽醌盐酸盐溶液（10mg/mL )， 在 60-65 进行载药。 孵育约 lh后， 使用凝胶排阻色谱测定包封效率。
2.一般方案 2
将磷脂如氢化大豆卵磷脂（ HSPC ) (或双软脂酸卵磷脂（ DPPC ) 或双肉豆寇酸卵磷脂（DMPC ) )、胆固醇 (Choi)按照（10: 1至 1: 1 ) 的摩尔比混合， 同时加入用聚乙二醇修饰的二硬脂酰磷脂酰乙醇胺 (DSPE-PEG), 其摩尔比为磷脂的 0.1%至 20%。 将上述混合物溶解 于有机溶剂如叔丁醇或者环己烷中，得到澄明溶液。之后采用常规技 术冷冻干燥得到均匀的冻干粉末。 将此冻干粉末用 （50-1000mM ) 的硫酸铵溶液、柠檬酸溶液或者过渡态金属的硫酸盐溶液（如硫酸镍 溶液）等在 水化，之后震荡约 lh，得到不均匀的多室脂质体。 之后使用微射流设备或者高压挤出设备降低脂质体的粒度。将所获得 的样品用浓度为 0.9%的 NaCl溶液稀释 200倍后， 用 NanoZS进行 检测。之后使用超滤装置移去空白脂质体外相的緩冲液， 以便形成跨 膜动力梯度。按照合适的脂药比，在空白脂质体中加入米托蒽醌盐酸 盐溶液（10mg/mL )， 在 60-65 进行载药。 孵育约 lh后， 使用凝胶 排阻色谱测定包封效率。
实施例 2
米托蒽醌脂质体 plm 60的制备
将 HSPC、 Choi和 DSPE-PEG2000按照 （3: 1: 1 ) 的质量比 混合， 在溶解于 95%叔丁醇中， 得到澄明溶液。 之后冷冻干燥得到 均匀的冻干粉末。 将此冻干粉末用 300mM的硫酸铵溶¾^ 60-65 水化， 之后震荡 lh， 得到不均匀的多室脂质体。 最终的磷脂浓度为 96mg/mL。 之后使用微射流设备降低脂质体的粒度。 将所获得的样 品用浓度 0.9%的 NaCl溶液稀释 200倍后， 用 NanoZS进行检测， 粒子的平均粒度约为 60nm， 主峰集中在 40-60nm之间。之后使用超 滤装置移去空白脂质体外相的硫酸铵，将外相置换成 250mM蔗糖及 50mM甘氨酸，以便形成跨膜硫酸铵梯度。按照脂药比 16: 1的比例， 在空白脂质体中加入米托蒽醌盐酸盐溶液（10mg/mL )， 在 60-65 进行载药。 孵育约 lh 后， 使用凝胶排阻色谱可证明包封效率约为 100%。 此处方被命名为 plm 60» 实施例 3
米托蒽醌脂质体 plm 85的制备
将 HSPC、 Choi和 DSPE-PEG2000按照 3: 1: 1的质量比混合， 在溶解于 95%叔丁醇中， 得到澄明溶液。 之后冷冻干燥得到均匀的 冻干粉末。 将此冻干粉末用 300mM的硫酸铵溶液在 60-65 水化， 之后震荡 lh， 得到不均匀的多室脂质体。 最终的磷脂浓度为 96mg/mL。 之后使用高压挤出的方法降低脂质体的粒度。 将所获得 的样品用 NaCl溶液稀释 200倍后， 用 NanoZS进行检测， 粒子的平 均粒度约为 85 nm。 之后使用超滤装置移去空白脂质体外相的硫酸 铵， 将外相置换成 250mM蔗糖及 50mM甘氨酸，以便形成跨膜硫酸 铵梯度。按照脂药比 16: 1的比例， 在空白脂质体中加入米托蒽醌盐 酸盐溶液（ 10mg/mL )， 在 60-65 进行载药。 孵育约 lh后， 使用凝 胶排阻色谱可证明包封效率约为 100%。 此处方被命名为 plm 85。 实施例 4
米托蒽醌脂质体 plmlOO的制备
采用与实施实例 3相同的方法制备米托蒽醌脂质体 plmlOOo 其 处方组成和 plm60及 plm85相同， 但是脂质体的粒度为 100nm。 实施例 5
米托蒽醌脂质体 plm60-dppc的制备
将 DPPC、 Choi和 DSPE-PEG2000按照质量比 3: 1: 1混合均 匀， 其他操作同实施实例 2。 此处方被命名为 plm60-dppc。 实施例 6
米托蒽醌脂质体 plm60-dmpc的制备
将 DMPC、 Choi和 DSPE-PEG2000按照质量比 3: 1: 1混合均 匀， 其他操作同实施例 2。 此处方被命名为 plm60-dmpc。 实施例 7
米托蒽醌脂质体 plm60-dmpc-0.1的制备
将 DMPC、 Choi和 DSPE-PEG2000按照质量比 3: 1: 0.1混合 均匀， 其他操作同实施实例 2。 此处方被命名为 plm60-dmpc-0.1。 实施例 8
阿審素脂质体 pld60的制备 在药物装载的过程中，将米托蒽醌置换为阿審素，其他操作同实 施实例 2。 此处方被命名为 pld60。 第二部分 不同的脂质体处方的释放情况 实施例 9
阿審素脂质体 pld60和米托蒽醌脂质体 plm60的释放的差异
在本例中， 米托蒽醌和阿審素是采用 pH梯度进行载药的。 如果 在释放介质中加入一定浓度的氯化铵，游离的氨分子就会借助梯度扩 散到脂质体的内相空间内， 从而升高内相的 pH值，使内相的质子化 的药物转变成中性的可以跨膜扩散的药物。这个过程同时又可以加快 脂质体内沉淀的溶解。药物从脂质体中释放的快慢，是受沉淀溶解和 膜通透性双重控制的。 释放的条件如下， 将脂质体用释放介质稀释 25倍。 释放介质分别含有 2、 10及 40mM的氯化铵， 等渗， pH为 7.4。 将稀释的脂质体装在透析袋中， 2mL的脂质体稀释液对 400mL 释放介质进行透析， 在 进行释放。 不同时间点取样进行分析， 一直取到 96小时。 将得到的数据进行回归。 pld60在含有 2、 10及 40mM的氯化铵的释放介质中， 药物释放的半衰期分别为 94.3、 31.9 和 11.2小时。 而对于 plm60而言， 在三种释放介质中均无明显的释 放。 由于这两个处方在脂质体组成和粒度上没有差异， 药物释放动力 学特性的不同可能应归因于药物特性的不同。阿審素和米托蒽醌均为 蒽环类抗生素，其差异在于阿審素含有一个生理 pH下可以解离的基 团 （pKa=8.15 )， 而米托蒽醌含有 2个生理 pH下可解离的基团。 这 个实施实例充分说明了象米托蒽醌这类含有多解离基团的药物，当使 用主动载药技术，会与反离子形成较复杂的沉积结构，从而显著延緩 药物在体外释放介质中的释放。 而对于单解离基团的药物如阿審素， 由于使用小粒度的处方，可能会使药物释放过快，在不含有血浆的释 放介质中也会较快释放。 实施例 10
不同粒度的米托蒽醌脂质体处方的释放行为
采用了两种不同的释放条件，比较了不同粒度的米托蒽醌脂质体 处方的释放。
释放条件 1: 将脂质体用释放介质稀释 25倍。 释放介质中含有 50%的人血浆， 采用葡萄糖调节等渗， pH为 7.4， 其他和实施实例 9 相同。 将得到的数据进行回归， 表明 plm60的释放半衰期为 56.4小 时， 而 plm85在上述条件下没有明显的释放。
释放条件 2: 释放介质中含有 50%的人血浆及 20mM氯化铵。 其他操作方法与实施实例 9相同。将得到的数据进行回归，结果发现 plm60的释放半衰期为 26.2小时， 而 plm85的释放半衰期为 36.7小 时。
本实施例充分说明了降低脂质体的粒度可以显著增加药物的释 放行为。 实施例 11
不同膜组成的米托蒽醌脂质体处方的释放行为
采用实施实例 9的释放条件， 结果发现， plm60-DPPC的释放半 衰期为 116小时， 而 plm60-DMPC的释放半衰期为 26小时，
plm60-DMPC-0.1的释放半衰期为 15小时。这个实验表明改用低 Tm 值的磷脂也可以加快药物的释放。 但是药物的释放行为的过渡增加， 会导致药物在体内的毒性过大， 在随后的实验中也可以发现这一点。 第三部分体内药代行为
实施例 12
PLM60在昆明小鼠体内的药代学行为及其与游离米托蒽醌药代对比 实验使用雄性体重约为 20g的昆明小鼠完成。不同剂量的米托蒽 醌通过尾静脉注入小鼠体内。 PLM的给药剂量为 1、 2和 4mg/kg， 游离米托蒽醌的给药剂量为 2mg/kg。 在不同的时间收集血浆样品。 血浆的处理方法和检测方法与文献（ Methods in enzymology Vol:391 176-185 )中报道的相似。 结果见表 1和附图 1。从图 1和表 1中可以 清楚看到，脂质体包裹可以显著的延长米托蒽醌的半衰期。 同剂量的 PLM60和 FM (游离米托蒽醌)相比，其在血液循环中的滞留时间是游 离药物的 32倍左右。 其 AUC值则约为游离药物的 3700倍。 同时以 AUC为因变量， 对剂量作图， 可以发现 PLM60在体内成线性代谢。 表 1： PLM60在昆明小鼠体内的药代学参数及其与游离米托蒽醌 (FM) 药代学参数对比
PLM60 PLM60 PLM60 FM
4 m /l^ 2 mg/l¾ 1 mg/kg 2 mg/kg 練 参数值 参数值 参数值 参数值
AUC 0-48(mg/L*h) 1451.666 728.398 452.709 0.198
AUC 0-∞(mg/L*h) 1654.543 892.437 503.078 0.199
AUMC 0-48 21838.034 12050.681 7049.259 0.103
AUMC O-oo 36234.611 24686.917 10488.811 0.135
MRT 0-48(h) 15.043 16.544 15.571 0.517
MRT 0-∞(h) 21.900 27.662 20.849 0.675
Tmax(h) 0.083 0.083 0.250 0.083
C (mg L) 86.329 47.513 25.970 0.699 实施例 13
PLM60和 FM在荷瘤小鼠体内的组织分布
PLM60和 FM在荷瘤小鼠体内的分布有明显的差异。 实验中使 用的小鼠为 20g左右的昆明雄性小鼠。将 S-180肉瘤细胞按照 5 X 105 的比例接种于小鼠右侧腋下。 当肿瘤生长至 0.4 0.7g时经静脉内施 用药物， 在给药后一定的时间点处死小鼠，取各组织器官的样品， 测 定其中米托蒽醌的含量。所测定的组织包括心、肝、脾、肺、 肾、肠、 髓以及肿瘤。 实验结果表明， PLM60对于肿瘤组织具有非常明显的 靶向性。 表 2和图 2给出了较详细的实验数据。 PLM60和 FM在正常小鼠组织内分布的实验数据
PLM-60 FM
4 mg/iig 4 mg/iig 时间 SD 时间 ^g/g SD 心 1 4.01 0.38 1 5.385 0.679
4 3.39 0.38 4 3.517 0.952
8 3.48 0.64 8 3.197 0.357
16 2.83 0.57 24 2.943 0.549
24 2.06 0.48
肝 1 6.78 0.78 1 4.770 0.997
4 5.99 0.67 4 3.556 0.543
8 6.31 0.38 8 2.659 0.439
16 6.22 0.95 24 1.937 0.346
24 4.52 0.65
脾 1 4.66 1.37 1 4.044 0.414
4 4.36 0.67 4 4.460 0.494
8 4.78 1.70 8 3.774 2.676
16 7.56 2.13 24 7.752 2.469
24 5.91 1.00
肺 1 8.44 1.08 1 10.205 1.732
4 4.58 2.36 4 8.024 1.859
8 6.33 1.43 8 7.018 0.728
16 5.12 1.24 24 8.082 0.844
24 2.89 0.23
1 7.09 0.84 1 18.243 1.238
4 7.12 1.17 4 17.192 5.010
8 7.04 0.96 8 13.409 1.251
16 6.75 1.16 24 7.463 1.209
24 5.82 0.84
肠 1 1.66 0.66 1 1.532 0.309
4 2.33 0.66 4 2.140 0.655
8 2.34 0.64 8 2.551 1.204
16 2.42 0.51 24 3.936 1.625
24 2.25 0.32
髓 1 1.09 0.54 1 0.127 0.041 4 0.64 0.14
8 0.73 0.16
16 0.54 0.24
24 0.12 0.02
肿瘤 1 91.28 7.41 0.0614 0.0078
4 63.90 8.56 0.0133 0.0027 8 54.01 8.04
16 38.61 9.19
24 10.41 2.67
实施例 14
不同脂质体处方的药代学对比
所用的实验动物与实施例 12相同， 三种处方的给药剂量均为 4mg/kg, 通 i^C静脉注入小鼠体内。 对比图 3和表 3中的数据， 可 以发现： 当脂双层的组成发生显著的变化后，会明显的改变脂质体药 物在体内的药代学行为。 PLM60-DPPC， PLM60-DMPC-0.1 和 PLM60-DMPC这三个处方在体内的 MRT值分别为 14.22， 7.914和 10.123小时。 其中 PLM60-DPPC和 PLM60-DMPC相比， 其处方的 差异就在于使用的磷脂的烃链的长度不同，分别为 16和 14碳。酰基 链的长短的不同， 可以显著的影响磷脂双分子层的通透性。 其中 DPPC 的 Tm 值为 41 度， 而 DMPC 的 Tm 值为 23 度。
PLM60-DMPC-0.1以及 PLM60-DMPC这两个处方相比， 差异就在 于 PEG化的程度不同。 脂质体在血浆中的释放跟 2种因素有关， 一 是药物跨磷脂双分子层的释放，另外一种与脂蛋白和网状内皮系统的 吞噬有关系。 由于 PLM60-DMPC-0.1脂质体的 PEG化不完全， 所 以由于血浆成分引起的释放对其有较大影响。 表 3: 不同处方在小鼠体内的药动学参数的对比
PLM60-DPPC PLM60-DMPC-0.1
4 mg/kg 4 m /l^ 4 m /l^
練 参数值 参数值 参数值
AUC 0-48(mg L*h) 1506.710 174.849 337.641
AUC 0-∞(mg/L*h) 1581.242 175.705 344.134
AUMC 0-48 21425.274 1383.757 3417.981
AUMC O-oo 26235.613 1478.267 3818.856
MRT 0-48(h) 14.220 7.914 10.123
MRT 0-∞(h) 16.592 8.413 11.097
Tmax(h) 1.000 1.000 1.000
C賺 (mg/L) 81.976 19.853 39.115 第四部分 不同处方的毒性对比 实施例 15
PLM60和 FM的急性毒性的对比
取昆明小鼠 100只， 雄雄各半， 体重在 18-22克。 一共分成 10 组， 每组 10只， 雄雄各半。 前五组给与不同剂量的游离药物， 后五 组给予等剂量的脂质体药物。观察小鼠的体重变化情况，并记录小鼠 的死亡时间。 小鼠死亡后， 对其进行解剖， 并进行尸检。 游离药物组 和脂质体组的死亡情况请见表 4。 实验证明 PLM60的急性毒性远低 于 FM。
性及其与游离米托蒽醌对比
Figure imgf000023_0001
NA: 表示无数据 (即在观察期结束时小鼠未死亡）。 实施例 16
不同脂质体处方的急性毒性的对比
取 Balb/c种小鼠 90只， 全部为雄性， 体重在 18-22克。 一共分 成 9组， 每组 10只。 第一组给予游离的米托蒽醌， 剂量为 6 mg/kg。 其余 8组分别给予 PLM60, PLM60-DPPC, PLM60-DMPC-0.1和 PLM60-DMPC剂量分别为 6和 12 mg/kg。观察小鼠的体重变化情况， 并记录小鼠的死亡时间。 小鼠死亡后， 对其进行解剖， 并进行尸检。 游离药物组和脂质体组的死亡情况请见表 5。 实验证明不同处方的急 性毒性顺序如下： PLM60 < PLM60-DPPC < PLM60-DMPC-0.1〜 FM < PLM60-DMPC。 这个实验证明同时采用小单室处方， 并且改 变磷脂双分子层的组成为 Tm较低的磷脂， 如 PLM60-DMPC, 会进 一步加快药物的释放速度。从而在体内造成更大的毒性。但是值得注 意的是， 不完全 PEG化的处方的毒性反而要低于高 PEG化的处方。 这可能是由于 PLM60-DMPC-0.1的 PEG化程度不完全， 在血液循 环的过程中， 由于脂蛋白的作用和免疫系统的攻击， 药物和 PLM60-DMPC相比，药物提前释放，没有在重要的组织内发生突释。 从而降低了毒性， 但是其毒性勉强与游离药物相当。 表 5: 不同脂质体处方的急性毒性的对比 处方及剂量 小鼠存活天数
( mg/l¾ ) 1 2 3 4 5 6 7 8 9 10
FM-6 NA NA NA NA NA NA NA NA NA NA
PLM60-6 NA NA NA NA NA NA NA NA NA NA
PLM60-12 NA NA NA NA NA 10 NA NA NA 11
Plm60DPPC-6 NA NA NA NA NA NA NA NA NA NA
Plm60DPPC-12 10 10 12 11 NA NA NA 13 NA 14
Plm60-DMPC-6 4 NA NA 3 6 7 7 6 NA NA
Plm60DMPC-12 3 3 5 3 3 3 4 3 3 3
Plm60DMPC-0.1-6 NA NA NA NA NA NA NA NA NA NA
Plm60DMPC-0.1-12 10 12 10 12 10 10 10 11 10 10
NA: 表示无数据 (即在观察期结束时小鼠未死亡）。 实施例 17
不同粒度的脂质体处方的毒性的对比
使用体重为 18〜22克的雄性 C57小鼠比较 plm60, plm85和 plmlOO的毒性。给药剂量为 9 mg/kg， 结果发现在上述情况下， 三个 处方的导致小鼠的体重变化相当，证明在上述试验条件下，这三个处 方的毒性没有明显的差异。 而同等剂量的游离药组的体重下降超过 30%, 并且有 20%的小鼠死亡。 第五部分体内抗肿瘤的效果
实施例 18
PLM60和 FM对 S-180肉瘤的治疗效果的对比
选取接种 S180瘤细胞 7天的腹水型荷瘤小鼠，断颈处死，抽取 乳白色粘稠腹水， 以 RPMI 1640培养基稀释， 稀释后调整瘤细胞数 为 2.5 x 106个细胞 / ml。将瘤细胞悬液接种于体重 18〜22 g KM雄性 小鼠前肢腋下皮下组织， 接种体积为 0.2 ml， 含瘤细胞约 5 x l05个， 接种完成后， 剩余的瘤细胞悬液在光镜下计数， 活瘤细胞数 > 95%。 共接种 80只小鼠。
接种 7天后， 挑选肿瘤边界清楚、 瘤径约 5的小鼠 39只， 按瘤 大小将动物分层，再按体重随机分组。分为空白对照组 7只， PLM60 4、 6 mg/k 组、 米托蒽醌游离药 4、 6 mg/kg组各 8只。 分别按组进 行静脉给药。
给药后动物正常饲养， 用游标卡尺测定肿瘤直径， 每周测量 3 次，每次测量同时称鼠重。 肿瘤体积（ tumor volume, TV )的计算公式 为： V = 1/2 X a X b2 (其中 a、 b分别表示长宽）， 相据测量结果计 算出肿瘤体积。 接种后第 21天脱颈处死小鼠， 剖取瘤块并称重。 根 据公式计算肿瘤抑制率（％)， 肿瘤抑制率 = ( 1-给药组平均瘤重 /对 照组平均瘤重） xl00%。 实验结果采用 t检验。
表 6显示， 米托蒽醌脂质体 ( PLM60 ) 4、 6 mg/k 剂量组显 著抑制 S180 实体瘤的生长。
PLM60对 S180实体瘤瘤重的影响
Figure imgf000025_0001
a :与空白组相比， p < 0.01 实施例 19
PLM60和 FM对 L1210腹水模型的治疗效果
选取接种 7天的 L1210腹水瘤 BDF1 小鼠， 断颈处死， 无菌 ^下抽取乳白色粘稠腹水， 以 RPMI 1640培养基稀释， 稀释后调 整瘤细胞数为 2.5X106个细胞 / ml。 将瘤细胞悬液腹腔接种于 7~8 周龄 BDF1 雌性小鼠，接种体积为 0.2 ml，含瘤细胞约 5.0 x 105个， 接种完成后， 剩余的瘤细胞悬液在光镜下计数， 活瘤细胞数 >95%。
24小时后，按体重随机分成 8组，分别给予米托蒽醌游离药 2，
4和 8mg/kg; 以及米托蒽醌脂质体 ( PLM60 ) 2、 4、 6和 8 mg/kg 分别尾静脉注射给药， 给药容积为 20ml/kg。 给药后动物正常饲养， 每周 3次测量鼠重，观察和记录动物死亡时间， 计算生存天数。 试验 结果以动物平均生存天数 (即 mean survival time, )和中位生存时间 (即 median survival time,MST )来评价每组的生存时间。 实验观察 至接种后 60天。
经 SPSS 11.5统计软件分析， 各给药组与空白组相比均显著增加 动物的生存时间， 相同剂量的米托蒽醌脂质体 ( PLM60 ) ( 8 mg/kg ) 与游离药组相比疗效显著增强 （P < 0.05)。 实验结果见表 7。 表 7 L1210腹水瘤对 BDF1小鼠生存时间的影响 剂量 动物 死亡
生存时间 （ 95%可信区间） 生存率 组别 (mg/k 数 动物数
(%) 平均生存时间 中位生存时间
g) (N) (N)
9.62±0.40 9.00±0.25
空白组 -- 13 13 0
( 8.83 ~ 10.40 ) (8.51-9.49)
20.17±3.77 14.00±1.15
2 12 11 8.33
( 12.77~27.56) ( 11.74~16.26)
36.75±4.00 31.00±0.85
PLM60 4 12 9 25.00
( 28.92 ~ 44.58 ) ( 29.33 ~ 32.67 )
28.42±4.49 25.00±3.46
6 12 10 16.67
( 19.63~37.21 ) ( 18.21~31.79)
36.42±4.08 29.00±1.71
PLM60 2 12 9 25.00
(28.41~44.42) (25.65 ~32.35 )
57.55±1.60
4 11 2 81.82
( 54.40 ~ 60.69 ) 48.00±4.38
6 12 5 Nb 58.33
( 39.42 ~ 56.58 )
53.00±3.71
8 12 4 Nb 66.67
( 45.72 ~ 60.28 )
Nb： 在观察期 60天结束时仅有少数动物死亡， 不能计算中位生存时间。 实施例 20
PLM60和 FM对 L1210肝转移模型的治疗效果
选取接种 7天的 L1210腹水瘤 BDF1 小鼠， 断颈处死， 无菌 ^下抽取乳白色粘稠腹水， 以 RPMI 1640培养基稀释， 稀释后调 整瘤细胞数为 2.5 X 105个细胞 / ml。 将瘤细胞悬液静脉接种于 7-8周 龄 BDF1雄性小鼠， 接种体积为 0.2 ml， 含瘤细胞约 5.0 x 104个， 接种完成后， 剩余的瘤细胞悬液在光镜下计数， 活瘤细胞数 > 95%„ 共接种 62 只小鼠。
24小时后， 分组给药。 给药后动物正常饲养， 每周 3次测量鼠 重，每天观察和记录动物死亡情况， 计算生存天数。 实验观察至接种 后 60天。
实验结果显示， 空白组小鼠在接种后第 11-14天全部死亡； 米托 蒽醌游离药三个剂量组所有动物在接种后第 11-17天期间全部死亡； 米托蒽醌脂质体 PLM60 2 mg/k 剂量组所有动物在接种后第 15-29 天期间全部死亡， 6 mg/k 剂量组仅一只动物在接种后第 39天死亡， 8 mg/k 剂量组无动物死亡。
经 SPSS 11.5统计软件分析， 与空白组相比， 米托蒽醌游离药 6 mg/k 剂量组和脂质体各剂量组均显著增加动物的生存时间。相同剂 量的米托蒽醌脂质体与其游离药组相比， 均显著提高动物的生存时 间。 实验结果见表 8。 表 8 静脉接种 L1210对 BDF1小鼠生存时间的影响
Figure imgf000028_0001
Na ： 所有动物在观察期 60天结束时无死亡， 不能计算生存时间。
Nb : 在观察期 60天结束时仅有一只动物死亡， 不能计算中位生存时间。 实施例 21
不同粒度的米托蒽醌脂质体的处方对于 L1210腹水瘤的治疗效果 实验方案及数据处理的模式与实施实例 19相同。 一共设置了 5 个实验组， 分别为空白组， 游离药物组、 plm60， plm85和 plmlOO 组。 每组的给药剂量分别为 4mg/kg。 实验结果表明小粒度的脂质体 具有较好的治疗效果。 实验结果见表 9。 L1210腹水瘤对 BDF1小鼠生存时间的影响
Figure imgf000029_0001
Nb： 在观察期 60天结束时仅有少数动物死亡， 不能计算中位生存时间。 尽管此处已经描述了本发明的某些优选实施方案，但是这些实施 方案不应被理解为是对本发明范围的限制。 实际上， 除了本文所显示 和描述的以外，在阅读了本申请的公开内容之后，结合本领域的普通 知识，普通技术人员将十分清楚本发明的各种其它修饰、改变和变化， 它们均应涵盖在本发明的保护范围之内。本文引用的所有专利、公开 的专利申请和出版物均通过引用并入， 就像全文在本文中列出一样。

Claims

权 利 要 求
1、 一种脂质体制剂， 其粒度为约 30-80 nm， 并且在磷脂双分子 层中含有 Tm 高于体温的磷脂， 从而脂质体的相转变温度高于体温， 优选所述磷脂是磷脂酰胆碱、 氢化大豆卵磷脂 （HSPC )、 氢化蛋黄 卵磷脂、 双软脂酸卵磷脂 （DPPC )或双硬脂酸卵磷脂 （DSPC )或 者其任何组合。
2、 根据权利要求 1所述的脂质体制剂， 其中所述磷脂双分子层 中 Tm高于体温的磷脂占总磷脂含量的约 50-100 mol/mol%，优选约 55-95 mol/mol % , 更优选约 60-90 mol/mol %。
3、 根据权利要求 1的脂质体制剂， 其中可选地在所述磷脂双分 子层中含有其它磷脂，例如 Tm值不高于体温的磷脂，如双肉豆寇酸 卵磷脂 （ DMPC )等。
4、 根据权利要求 1的脂质体制剂， 其中可选地含有胆固醇， 其 含量为脂质体各成分总摩尔数的 2-60 mol/mol% , 例如 5-55 mol/mol%, 尤其是 10 - 50 mol/mol%， 特别地 15-45 mol/mol%， 更 特别地 20-40 mol/mol%。
5、根据权利要求 1的脂质体制剂，其中可选地包含其他的辅料， 例如进一步修饰脂质体表面特征的辅料，如用亲水性聚合物修饰的脂 类物质， 其可选自例如聚乙二醇修饰的二硬脂酰磷脂酰乙醇胺 (DSPE-PEG)、 聚乙二醇修饰的二硬脂酰磷脂酰甘油 (DSPG-PEG)、 聚乙二醇修饰的胆固醇 (chol-PEG)、聚维酮修饰的二硬脂酰磷脂酰乙 醇胺（DSPE-PVP)、 聚乙二醇修饰的二硬脂酰磷脂酰甘油 (DSPG-PVP). 或者聚乙二醇修饰的胆固醇 (chol-PVP)或者其组合， 优选其存在量以摩尔数计是磷脂的 0.1-20 mol/mol %， 例如 0.3-18 mol/mol%、 0.5-15 mol/mol%、 0.8-12 mol/mol%、 1-10 mol/mol%、 2-8 mol/mol%、 2.5-7 mol/mol%或者 3-6 mol/mol%等。 （其含量优选 地为脂质体的约 1-30 mol/mol %、 3-28 mol/mol%、 5-25 mol/mol%或 者 8-20 mol/mol%, 尤其是 10-20 mol/mol% )。
6、根据权利要求 1的脂质体制剂，其粒度为 35-75 nm，优选 40-70 nm， 尤其是 40-60 nm。
7、 根据权利要求 1的脂质体制剂， 其中包含氢化大豆卵磷脂、 胆固醇和聚乙二醇修饰的二硬脂酰磷脂酰乙醇胺，其质量比为 3:1:1， 优选所述聚乙二醇修饰的二硬脂酰磷脂酰乙醇胺是聚乙二醇 2000修 饰的二硬脂酰磷脂酰乙醇胺。
8、一种脂质体药物制剂，其在权利要求 1 - 7中任一项所述的脂 质体制剂中包含药物， 优选多价离子药物。
9、 根据权利要求 8所述的脂质体药物制剂， 其特征在于药物具 有两个或两个以上的解离常数 pKa在 4.5〜9.5之间的可解离基团， 优选解离常数 pKa在 5.0-9.5之间， 更优选在 5.5-9.5之间，特别优选 在 6.0-9.0之间， 尤其是 6.5-9.0之间。
10、根据权利要求 8所述的脂质体药物，其中所述多价离子药物 为抗癌药， 例如米托蒽醌、 长春新碱、 长春瑞滨、 长春碱或者其任意 组合， 优选米托蒽醌。
11、根据权利要求 8 - 10中任一项所述的脂质体药物制剂，其中 以药物制剂的总重量计， 所述药物的含量为 0.1-50wt%， 优选 0.5-40wt%，更优选 1—— 35wt%，特别优选 3-30wt%，或者 5-25wt%， 或者 8-20wt%。
12、根据权利要求 8 - 10中任一项所述的脂质体药物制剂，其中 任选地含有一种或多种其它药物成分， 和 /或药学上可接受的载体和 / 或赋型剂。
13、根据权利要求 8所述的脂质体药物制剂，其中所述脂质体内 含有反离子， 优选多价反离子， 例如有机酸根， 比如选自以下饱和或 不饱和有机酸的酸根： 柠檬酸、 酒石酸、 富马酸、 草酸、 丙二酸、 琥 珀酸、 苹果酸， 以及马来酸等， 无机酸根比如硫酸根、磷酸根或者氨 基酸、比如胱氨酸的离子化形式，优选柠檬酸根、硫酸根或者磷酸根。
14、 根据权利要求 13所述的脂质体药物制剂， 其中所述多价反 离子具有两个或两个以上与活性药物成分相反的电荷。
15、根据权利要求 8 - 10中任一项所述的脂质体药物制剂，其中 所述脂质体中包含磷脂酰胆碱、 氢化大豆卵磷脂、 氢化蛋黄卵磷脂、 双软脂酸卵磷脂或者双硬脂酸卵磷脂， 或者其组合。
16、 制备权利要求 8 - 15中任一项所述脂质体药物制剂的方法， 其包括如下步骤： （1) 利用 Tm值高于体温的磷脂以及可选地其它磷 脂和 /或胆固醇， 制备脂质体； 和 (2)将感兴趣的药物、 特别是多价离 子药物包封在脂质体内。
17、一种治疗患者中疾病、特别是肿瘤的方法， 所述方法包括给 需要该治疗的患者施用根据权利要求 8至 15中任一项的脂质体药物 制剂。
18、 根据权利要求 17的方法， 其中还包括对肿瘤患者联合应用 热疗法， 例如放射热疗法。
19、 权利要求 1 - 7中任一项所述的脂质体制剂或者权利要求 8 - 15 中任一项所述的脂质体药物制剂在制备用于治疗肿瘤患者的药 物中的用途。
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